System and method for the deposition, detection and identification of threat agents using a phase mask

ABSTRACT

A system method for depositing a sample of a threat agent is deposited onto a substrate. The threat agent is illuminated via an illumination source along an optical path with a plurality of photons to thereby produce photons transmitted, reflected, emitted or Raman scattered by the threat agent. An optical system collects elastic scatter photons produced by the threat agent and at least one of photons transmitted, reflected, emitted or Raman scattered by the threat agent, wherein said illumination source is located along an optical path, and said substrate is located along a plane wherein the optical path is at an angle other than 90° with respect to the substrate plane. The depth of field of the optical system is extended by passing at least one of the following through a phase mask: elastic scattered photons, and photons transmitted, reflected, emitted or Raman scattered by the threat agent.

RELATED APPLICATIONS

This application claims the benefit of U.S. Patent Application No.60/651,375 filed Feb. 9, 2005 entitled Development of a Biological RamanElectrostatic Detector Identifier (BioREDI) Sensor.

FIELD OF DISCLOSURE

This application relates generally to systems and methods for detectingand identifying hazardous agents.

BACKGROUND

Deployment of threat agents poses significant threats to both human andeconomic heath. This threat is compounded by a limited ability to detectdeployment of the agents. Prior art detection strategies rely onseparate instrumentation for detection and identification of the threatagent. Conventional means of detecting airborne matter includerelatively non-specific optical and spectroscopic methods, includinglaser scattering, ultraviolet laser induced fluorescence (UV-LIF) andlaser induced breakdown spectroscopy (LIBS). Conventional means toidentify a threat agent include wet chemical methods or spectroscopicmethods. Reagent-based identification of biological threat agentsincludes methods such as specific antibodies, genetic markers andpropagation in culture. While highly specific, these identificationmethods are time-consuming, labor-intensive and costly. Spectroscopicmeans, for identification, provide an alternative to reagent-basedidentification methods and include mass spectrometry, infraredspectroscopy, Raman spectroscopy, and imaging spectrometry. Massspectrometry is limited by sensitivity to background interference.Infrared spectroscopy exhibits low sensitivity. Raman spectroscopy canbe implemented in several different configurations, including normalRaman spectroscopy, UV resonance Raman spectroscopy, surface enhancedRaman spectroscopy (SERS) and non-linear Raman spectroscopy. Whilenormal Raman spectroscopy has demonstrated adequate sensitivity andspecificity for detection of airborne matter, other forms of Ramanspectroscopy suffer from inadequate sensitivity, specificity orsignature robustness. Prior art imaging spectroscopy is limited by theneed to switch from a broad band light source, for optical imaging, to asubstantially monochromatic light source for spectroscopic imaging. Thisresults in a signification time period between detection andidentification during which time the sample may degrade.

The present disclosure describes a reagent free sensor using Ramanspectroscopy and Raman imaging spectroscopy to detect and identify asample simultaneous with sample deposition. The system and methods ofthe present disclosure may operate in either a trigger mode or anidentification mode. The present disclosure describes an approach thatreduces system and method complexity by using a single illuminationsource.

SUMMARY

The present disclosure provides a system and method for depositing asample of a threat agent onto a substrate. The threat agent is detectedand identified substantially coincident in time with the deposition ofthe sample of the threat agent onto the substrate. Optionally, a fiberarray spectral translator that converts a non-linear field of view of aportion of the substrate containing the sample to a curvilinear mapcoupled to an entrance slit of a spectrometer is used to collectspatially-resolved Raman spectra suitable for identifying the threatagent.

In one embodiment, the threat agent deposited on the substrate isilluminated, via a single illumination source, with a plurality ofphotons to thereby produce elastic scattered photons and Raman scatteredphotons. Identifying the threat agent may be performed by analyzing theelastic scattered photons, produced by the threat agent on thesubstrate, using elastic scattering imaging to form an image of thethreat agent, and analyzing the Raman scattered photons, produced by thethreat agent on the substrate, using Raman spectroscopy. A Ramanspectrum may be compared to at least one reference Raman libraryspectrum to identify the threat agent. Analysis of the Raman scatteredphotons may further include generating Raman spectra anywhere within theRaman shift range of about 0 cm⁻¹ to about 3500 cm⁻¹ at a spectralresolution less than 20 cm⁻¹. Analysis of the Raman scattered photonsmay further include generating multiple spatially independent imagechannels simultaneously within a Raman shift of about 0 cm⁻¹ to about3500 cm⁻¹ at a full spectral resolution less than 20 cm⁻¹.

In one embodiment, analysis of the elastic scattered photons produced bythe threat agent includes automatically focusing the image of the threatagent on the substrate using one of the following: a CMOS detector, aCCD detector or a high frame rate digital detector, in combination witha feedback control mechanism. Analysis of the Raman scattered photonsalso may include passing the Raman scattered photons through a deviceselected from the group consisting of a tunable filter, a band passfilter, a liquid crystal tunable filter, an interferometer, an acoustooptic tunable filter and a dispersive optical device, to produce theplurality of spatially resolved Raman spectra. Analysis of the Ramanscattered photons may also include passing the Raman scattered photonsthrough one of the following: a line scan spectrometer; a multi-pointspectrometer; a single point spectrometer; and area imagingspectrometer.

In one embodiment, the elastic scattered photons and the Raman scatteredphotons are collected using an optical system, wherein said illuminationsource is located along an optical path, and said substrate is locatedalong a plane wherein the optical path or the deposition apparatus is atan angle other than 90° with respect to the substrate plane.

In one embodiment, the system and method for depositing and identifyingthe threat agent operates in a trigger mode that detects a presence orabsence of the threat agent, and an identification mode that identifiesthe threat agent. The trigger mode may use a trigger time period and theidentification mode may use an identification time period, and whereinthe trigger time period is less than the identification time period. Inone embodiment, the identification mode is initiated upon detecting thepresence of the threat agent in the trigger mode. An additional amountof the threat agent may be accumulated during operation in theidentification mode. In one embodiment, the identification mode may beinitiated substantially simultaneous upon detecting the present of thethreat agent in the trigger mode.

Depositing the threat agent onto the substrate may be accomplished usingultrasonic deposition, electro spray and inertial impaction of thethreat agent onto the substrate. In one embodiment, depositing thesample of the threat agent onto the substrate includes depositing atleast 50 particles onto the substrate. Depositing the sample of thethreat agent onto the substrate may include collecting air from aconfined environment or an outside environment.

The threat agent may be hazardous agent comprising a bacterium, virus,protozoan, biological toxin, fungus, a chemical agent, a radiologicalmaterial and an explosive material and/or may be an airborne particulatematter or aerosol matter.

In accordance with a further aspect, the present disclosure provides asystem and method for depositing a sample of a threat agent onto asubstrate. A single illumination source illuminates the threat agentdeposited on the substrate with a plurality of photons to therebyproduce elastic scattered photons and Raman scattered photons. Thethreat agent on the substrate is identified. The system and methodoperate in a trigger mode that detects the presence or absence of thethreat agent, and an identification mode that identifies the threatagent. Optionally, a fiber array spectral translator that converts anon-linear field of view of a portion of the substrate containing thesample to a curvilinear map coupled to an entrance slit of aspectrometer which is used for identifying the threat agent.

Deposition of the sample of the threat agent onto the substrate mayoccur prior to identification of the threat agent on the substrate. Abackground level of the substrate may be identified before deposition ofthe sample of the threat agent onto the substrate. In this embodiment,identifying the threat agent on the substrate occurs substantiallycoincident in time with or after the depositing of the sample of thethreat agent onto the substrate.

In accordance with a further aspect, the present disclosure provides asystem and method for depositing a sample of a threat agent onto asubstrate. The deposition of the threat agent onto the substrate isvisually observed by analyzing the elastic scattered photons produced bythe threat agent using elastic scatter imaging to form an image of thethreat agent on the substrate, wherein depositing of the threat agent issubstantially coincident in time with visually observing of thedeposition of the threat agent. Analyzing the elastic scattered photonsproduced by the threat agent may include automatically focusing theimage of the threat agent on the substrate using a CMOS detector, a CCDdetector or a high frame rate digital detector, in combination with afeedback control mechanism. The elastic scattered photons may becollected via an optical system, wherein the optical system ispositioned relative to the substrate and moved relative to the positionof the substrate to focus the image of the threat agent on thesubstrate. Image contrast in the image of the threat agent on thesubstrate may be improved by removing an interference pattern of theillumination source via mode scrambling and frame averaging. Visuallyobserving deposition of the threat agent onto the substrate is performedwithout a spectrometer.

In accordance with a still further aspect, the present disclosureprovides a system and method for depositing a sample of a threat agentonto a substrate. A single illumination source illuminates the threatagent on the substrate with a plurality of photons to thereby produceelastic scattered photons. Deposition of the threat agent onto thesubstrate is visually observed by analyzing the elastic scatteredphotons produced by the threat agent using elastic scatter imaging toform an image of the threat agent on the substrate.

In accordance with a still further aspect, the present disclosureprovides a system and method for identifying a sample of a threat agentthat is deposited onto a substrate. A first optical collection devicecollects at least one of the following: elastic scattered light producedby the threat agent, and Raman scattered light produced by the threatagent. A second optical collection device collects Raman scattered lightproduced by the threat agent, wherein the second optical collectiondevice comprises a two dimensional non-linear array of optical fibersdrawn into a one dimensional fiber stack that converts a non-linearfield of view into a curvilinear map, wherein the curvilinear fiberstack is coupled to an entrance slit of a Raman spectrometer. The threatagent deposited on the substrate is identified using Raman spectroscopy.

In accordance with yet a further aspect, the present disclosure providesa system and method for identifying a sample of a threat agent that isdeposited onto a substrate. The threat agent is illuminated via anillumination source with a plurality of photons to thereby producephotons transmitted, reflected, emitted or Raman scattered by the threatagent. An optical system collects elastic scatter photons produced bythe threat agent and at least one of photons transmitted, reflected,emitted or Raman scattered by the threat agent, wherein saidillumination source is located along an optical path, and said substrateis located along a plane wherein the optical path or the depositionapparatus is at an angle other than 90° with respect to the substrateplane. The depth of field of the optical system is extended by passingat least one of the following through a phase mask: elastic scatteredphotons, and photons transmitted, reflected, emitted or Raman scatteredby the threat agent.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are included to provide furtherunderstanding of the disclosure and are incorporated in and constitute apart of this specification, illustrate embodiments of the disclosureand, together with the description, serve to explain the principles ofthe disclosure.

In the drawings:

FIG. 1 illustrates a system used in connection with the presentdisclosure;

FIG. 2 illustrates a device used in the system of this disclosure;

FIGS. 3A and 3B illustrate an elastic scatter image produced by thesystem and methods of the present disclosure;

FIGS. 4A, 4B, 4C and 4D illustrate Raman imaging analysis using thesystem and methods of the present disclosure; and

FIG. 5 illustrates the estimated sensitivity of detection andidentification using the system and methods of the present disclosure.

DESCRIPTION OF THE EMBODIMENTS

Reference will now be made in detail to the embodiments of the presentdisclosure, examples of which are illustrated in the accompanyingdrawings. Wherever possible, the same reference numbers will be usedthroughout the drawings to refer to the same or like parts.

FIG. 1 illustrates system 100 which may be used to carry out the methodsof the present disclosure. System 100 includes a deposition means 101and a detector means 113. The deposition means may include an air intakeport 104, which is open to the surrounding environment 102, a collector106, a concentrator 108, a sample 109, a deposition substrate 110 (e.g.,a compact disc), substrate plane 111 and a substrate positioningmechanism 112. The identification means 113 comprises a first opticalsystem 114, an optional phase mask 115, a beam splitter 116, a secondoptical system 117, an optical path 119, an elastic scattering imagedetector 118, an illumination source 122, a dichroic mirror 120, amirror 124, a spectroscopic detector 126, spectrometer 127 and aprocessor 128 having a spectral library 130.

As illustrated in FIG. 1, the sample is collected from the surroundingenvironment 102 and then concentrated. The sample may comprise airborneparticulate matter or aerosol matter. The surrounding environment 102includes a confined environment and an outside environment. The confinedenvironment includes a building, storage container, plane, train orother mass transportation vehicle and a human respiratory system. Tocollect air from a confined environment, system 100 is connected to theair conditioning or heating system of a building, vehicle or storagecontainer that circulates air to the confined environment.

The sample collected and identified by system 100 includes a threatagent. The threat agent comprises a hazardous agent and includes abacterium, virus, protozoan, biological toxin, fungus, a chemical agent,a radiological material and an explosive material. The bacteriumincludes Anthrax, Bacillus, Streptococcus, Staphylococcus, Escherichia,Erwinia, and Pseudomonas. The virus includes a pathogenic virus selectedfrom smallpox, influenza and E. bola viruses. The biological toxinincludes ricin. The hazardous substance is any substance that may causedisease, injury, discomfort, pain, or death to an animal such as ahuman.

The sample may be collected and concentrated using a variety of devices.In one embodiment, the sample is collected using an aerosol collector incombination with a virtual impactor which eliminates air andconcentrates the sample. In second embodiment, the sample is collectedusing an aerosol collector in combination with a liquid concentrator.This collection and concentration process takes place on the order of afraction of a second to minutes depending on the velocity of thecollecting air. The concentrated sample is subsequently deposited ontothe surface of the substrate. The sample may be deposited onto thesurface of the substrate using inertial impaction, ultrasonicdeposition, and electro spray deposition.

In one embodiment, ultrasonic deposition is used to deposit the sampleonto the substrate. In one embodiment, a wet walled cyclone collectormay be used to collect aerosol and particulate matter. Theanalyte-containing fluid, which can be connected to a reservoir,including a water storage tank, can be used to provideanalyte-containing fluid to the ultrasonic nozzle liquid inlet port. Theultrasonic nozzle may also contain a compressed air inlet to focus thedeposition of the liquid input onto the substrate surface. Theultrasonic spray device may be used to perform a plurality of sprayapplications over the same spatial location to increase the analyteconcentration in a desired field of view. In one embodiment, ultrasonicspray devices such those manufactured by Sono-Tek Corporation of Milton,N.Y may be used for implementing the present disclosure.

The deposition device deposits a plurality of sample particles 109 ontothe substrate 110. In one embodiment, at least 1 sample particle isdeposited onto the substrate. In another embodiment, at least 50 sampleparticles are deposited onto the substrate. In another embodiment, atleast about 50-250 sample particles are deposited onto the substrate. Inanother embodiment, at least about 250-2500 sample particles aredeposited onto the substrate. In another embodiment, at least about2500-10,000 sample particles are deposited onto the substrate. Inanother embodiment, at least about 10,000-100,000 sample particles aredeposited onto the substrate. In another embodiment, at least about100,000-1,000,000 sample particles are deposited onto the substrate.

With further reference to FIG. 1, system 100 uses a single illuminationsource 122, directed along an optical path 119, to illuminate the samplewith a plurality of photons to thereby produce elastic scattered photonsand photons transmitted, reflected, emitted or Raman scattered by thesample. In one embodiment, the illumination source illuminates thesample with a plurality of photons to produce elastic scattered photonsand Raman scattered photons. The illumination source includes a lowpower laser. Low power lasers manufactured by Coherent Inc, Santa Clara,Calif. or the Spectra-Physics Division of Newport Inc., Mountain View,Calif. are suitable. In one embodiment, the optical path 119 of theillumination source 122 is at an angle other than 90° from the plane 111defined by the two dimensional substrate 110. In another embodiment thedeposition means 101 is at an angle other than 90° from the plane 111defined by the two dimensional substrate 110.

With further reference to FIG. 1, system 100 has a first optical system114. In one embodiment, the optical system 114, collects elasticscattered photons, produced by the sample. In a second embodiment, theoptical system 114, collects elastic scattered photons and Ramanscattered photons produced by the sample. In a third embodiment, theoptical system 114, collects elastic scattered photons, and at least oneof photons transmitted, reflected, emitted or Raman scattered producedby the sample.

With further reference to FIG. 1, system 100 may include a phase mask115. The phase mask 115 will be used to extend the depth of field of theoptic system by passing through the phase mask 115 at least one ofelastic scattered photons and photons transmitted, reflected, emitted orRaman scattered produced by the sample.

One embodiment of the system 100 may include a second optical system, afiber array spectral translator (“FAST”). With reference to FIG. 2, theFAST system 200 includes a first lens 206, an illumination source 208, afirst filter 210, a second filter 212 a second lens 214, a first end ofa fiber bundle 216 and a second end of the fiber bundle 218 which isconnected to a spectrometer 220. The first lens 206 acts as a collectinglens which focuses the illumination source onto the sample 204 andcollects all photons, other than elastic scattered photons, atwavelengths other than laser wavelength; this includes photons emittedor Raman scattered by the sample. Photons transmitted or reflected bythe sample will have the same wavelength as the laser and will beblocked by filter element 212. Lens 206 collimates the photons producedby the sample projecting the photons into infinity. The second lens 214is used in combination with the first lens 206 to form images at thefinal focal plane of the second lens 214. The first end of the fiberbundle 216 is comprised of a two dimensional non-linear array of fiberbundles. The second end of the fiber bundle 218 is comprised of acurvilinear array of fiber bundles wherein curvilinear may include astraight line as well as a curved line configurations.

In one embodiment of the present disclosure, system 100 utilizes anelastic scatter imaging detector 118 to visually observe deposition andform an image of the sample on the substrate by analyzing elasticscattered photons produced by the sample. The image is used to assesssample deposition density, morphology and focusing. In one embodiment,the elastic scatter imaging, to visually observe deposition, is used inthe absence of an imaging spectrometer. In a second embodiment, system100 employs an elastic scatter imaging detector 118 in combination witha spectrometer 127 for identification of the sample. In one embodiment,the elastic scatter imaging detection and spectrometric identificationare performed simultaneously, using a single low power illuminationsource for identification of the sample.

With further reference to FIG. 1, the elastic scatter imaging detector118 is comprised of one of the following of a CMOS detector, a CCDdetector and a high frame rate digital detector. The system uses thedetector 118 in combination with a feedback and control mechanism toautomatically focus the sample under the collection optic. In oneembodiment, a light gathering objective of the optical system 114 ismoved relative to the position of the substrate to focus the image ofthe sample on the substrate. The spectrometer 127 could employ detectorssuch as CCDs, CMOS, CIDs (charge injection device), diode arrays,photomultiplier tube (PMT), PMT array, avalanche photodiode.

In one embodiment of the present disclosure, the elastic scatter imageof the sample is collected on the detector and mode scrambling and frameaveraging are used to improve the image contrast by removing theinterference pattern of the illumination source producing the finalimage. FIGS. 3A and 3B illustrate an elastic scatter image of humanepithelial cells obtained by one embodiment of the system of the presentdisclosure. A low power laser source illuminated the sample of humanepithelial cells to generate the elastic scatter image of the samplewhich produces a high cross section elastic scatter image signal.However, the elastic scatter image is typically masked by the presenceof a laser interference pattern. The laser interference pattern isremoved by mode scrambling and frame averaging. As illustrated in FIG.3A, the human epithelial cells are not readily observable due to thepresence of a laser interference (i.e. speckle) pattern masking thepresence of the inherently low contrast cellular object within the fieldof view of the 20× objective employed to capture the image. Asillustrated in FIG. 3B, the cell is readily observable by using a fiberoptic to scramble the modes traversing the fiber optic, whicheffectively makes the monochromatic laser light incoherent. A pluralityof images is time averaged and the cellular image contrast is enhancedby effective suppression of the coherent laser speckle pattern. Toobtain the image in FIG. 3B, ten images were each collected at 30 framesper second.

In another embodiment of the present disclosure, the elastic scatterimage of the sample is obtained by collecting the scattered photonsusing an optical system, wherein the optical path 119 of theillumination source 122 or the deposition means 101 is at an angle otherthan 90° from the plane 111 defined by the two dimensional substrate110. In one embodiment, this is achieved by operating the objective,which collects the elastic scattered photons, at 60° off axis from theplane of the substrate. This embodiment produces a finite rectangularregion of interest in focus but this region of interest will be smallerthan the objective field of view. In order to compensate for the out offocus regions of interest, extended depth of field (“EDF”) opticalcompensation may be used to image over the full field of view of themicroscope objective. To implement EDF, a phase mask will be located inthe elastic scatter image collection optical path to modify theincoherent optical system in such a way that the point spread function(“PSF”) is insensitive to misfocus, while forming an optical transferfunction (“OTF”) that has no regions of zero values within its passband. Because the OTF has no regions of zeros, digital processing can beused to “restore” the sampled intermediate image. Further, because theOTF is less sensitive to misfocus, the same digital processing restoresthe image for all values of misfocus. This combined optical/digitalsystems produces a PSF that is comparable to that of the diffractionlimited PSF, but over a far larger region of focus. Typically, an 8×increase in the DOF is achievable.

In another embodiment, system 100 utilizes a spectrometer 127 incombination with an elastic scatter imaging detector 118 to identify thesample. The elastic scattered photons, produced by the threat agent, areanalyzed using elastic scatter imaging to produce an image of the sampleon the substrate. In one embodiment, a spectrometer is used to analyzeat least one of photons transmitted, reflected, emitted or Ramanscattered by the sample, using spectroscopy. In another embodiment, aspectrometer is used to analyze at least one of photons transmitted,reflected, emitted or Raman scattered by the sample, using spectroscopicimaging to produce a plurality of spatially resolved spectra. Thespectrometer may operate in one or more of the following spectralranges: the ultraviolet (UV), visible, near infrared, and mid-infrared.The spectrometer may operate to collect images based on the followingdetection modalities: UV, visible, near-IR or mid-IR absorption imagingin either transmission or reflectance modes; Raman scatter imaging;fluorescence; photoluminescence; chemiluminescence; andelectroluminescence imaging. The spectrometer may operate in conjunctionwith polarized light microscopy and/or differential interferencecontrast imaging. Photons transmitted, reflected, emitted or Ramanscattered by the sample, are passed through a filter to produce aplurality of spatially resolved spectra. The filter may be a tunablefilter, a band pass filter, a liquid crystal tunable filter, aninterferometer, an acousto optic tunable filter or a dispersive opticaldevice. The photons transmitted, reflected, emitted or Raman scatteredmay be passed through a spectrometer which may be a line scanspectrometer; a multi-point spectrometer; a single point scanspectrometer or area imaging spectrometer. In one embodiment, thespectrometer may be used in an imaging mode to produce a plurality ofspatially resolved spectra arising from the sample volume illuminated bythe illumination source. In another embodiment, the spectrometer may beused in a non-imaging mode by summing all of the spectra collected inthe imaging mode to form a composite spectrum arising from the samplevolume illuminated by the illumination source. In another embodiment,the spectrometer may be used in a non-imaging mode to collect acomposite spectrum arising from the sample volume illuminated by theillumination source.

In one embodiment, the spectrometer includes a Raman imagingspectrometer that analyzes Raman scattered photons produced by thesample. In one embodiment, Raman imaging spectrometer generates Ramanspectra anywhere within the range of Raman shift of 0-3500 cm⁻¹, at afull spectral resolution of less than 20 cm⁻¹. In one embodiment, theRaman imaging spectrometer simultaneously captures multiple spatiallyindependent spectra, up to 1000, within the range of Raman shift of0-3500 cm⁻¹ at a full spectral resolution of less than 20 cm⁻¹. In oneembodiment, the spectrometer comprises a Raman line imagingspectrometer. In another embodiment, the spectrometer comprises adispersive Raman line imaging spectrometer.

In one embodiment, the sample is identified using a spectrometer incombination with a fiber array spectral translator. With reference toFIG. 4, a plurality of Raman spectra were collected on a ChemImageFALCON II Raman microscope from a physical mixture of a sample comprisedof ovalbumin (“Ova”) and diesel soot (“DS”) The spatially resolved Ramanspectra collected from the sample were mapped to the entrance slit ofthe FALCON II's dispersive Raman spectrometer, using a coherent fiberoptic bundle (FAST). Using FAST, a fiber the circular field of view ofthe image is converted to a curvilinear shape that is mapped to theentrance slit of the spectrometer. The benefits of this approach includerapid collection of the full Raman image spectral hypercube without theneed for spatial or spectral scanning mechanisms. Because multiple Ramanspectra can be captured within a wide field of view without the need toreposition the laser, fluorescence photo bleaching time can beminimized. In addition, the spatially resolved Raman spectra map thelocal heterogeneity of the sample mixture which enables target testingbased spectral unmixing. As a result, sample mixtures are analyzed inreal time if sufficient single to noise reduction is achieved. FIG. 4Aillustrates the optical image of Ova/DS under 100× objectivemagnification. FIG. 4B illustrates a dispersive Raman spectrumrepresenting the average response of the mixture sample, and FIG. 4Cillustrates seven spatially resolved dispersive Raman spectra collectedfrom the sample using FAST. FIG. 4D illustrates the dispersive Ramanspectral image collected at the focal plane of the spectrometer. Theseven independent spatial channels images along the y axis and the Ramanspectrum along the x axis of the focal plane are clearly visible.

In yet another embodiment, system 100 will utilize a phase mask 115 toextend the depth of field of the optical system. In this embodiment, asample 109 will be deposited onto a substrate 110. The sample will beilluminated via an illumination source 112 along an optical path 119with a plurality of photons producing elastic scattered photons and atleast one of photons transmitted, reflected, emitted or Raman scatteredby the sample. The sample may comprise a threat agent. An optical systemwill collect the elastic scatter photons produced by the sample and atleast one of photons transmitted, reflected, emitted or Raman scatteredby the threat agent. The illumination source 122 will be located alongthe optical path 119, and said substrate 110 will be located along aplane 111 wherein the optical path 119 is at an angle other than 90°with respect to the substrate plane 111. In another embodiment, thedeposition means 101 will be located at an angle other than 90° withrespect to the substrate plane 111. The depth of field of the opticalsystem will be extended by passing at least one of the following throughthe phase mask 115: elastic scattered photons, and photons transmitted,reflected, emitted or Raman scattered by the threat agent. The samplewill be identified based at least in part on the output of the phasemask 115, by (1) analyzing the elastic scattered photons using elasticscattering imaging 118 to form an image of the sample; and (2) analyzingat least one of photons transmitted, reflected, emitted or Ramanscattered by the sample using spectroscopic imaging 126 to produce aplurality of spatially resolved spectra. The photons transmitted,reflected, emitted or Raman scattered will be passed through a filter toproduce a plurality of spatially resolved spectra. The sample depositionand identification will occur: substantially coincident in time witheach other; deposition prior to identification; backgroundidentification followed by deposition which may be substantiallycoincident or prior to identification.

The method of the present disclosure provides for deposition, visualobservation and identification of the sample. In one embodiment, thesample is visually observed and identified substantially coincident intime with the deposition of the sample onto the surface of thesubstrate. The deposition process takes place over a fraction of asecond from the deposition of the first particle to the deposition of aparticle density sufficient to generate a measurable signal whichpermits the detection and identification of the sample, via the imagingspectrometer. The system of this disclosure has demonstrated that ameasurable signal, sufficient to visually observe and identify thesample via Raman imaging spectroscopy, may be observed in as short as 10seconds after being illuminated by the illumination source. In oneembodiment, the particle density, required for observation andidentification, is 20 sample particles deposited on the substrate, inthe detector field of view for a five minute identification mode. Inanother embodiment, the sufficient particle density may be as small as 1particle of a sample deposited on the substrate. In the case ofbioaerosols, the sample occurs as individual spores or cells oraggregates of cells. A single cell generates a measurable Raman signalwherein the time period for the Raman process, to generate a measurablesignal, is initiated within femtoseconds after being illuminated by theillumination source. Therefore, instantaneous deposition, observationand identification of a single cell are envisioned.

System 100 is used in a method of the present disclosure provides fordeposition and identification of a threat agent. In a preferredembodiment, the particle sample deposition is substantially coincidentwith identification of the particle sample. For the purposes of thisapplication, the term substantially coincident means that sampledeposition occurs on approximately the same time period as sampleidentification, ideally without the requirement that the samplesubstrate be re-positioned relative to the deposition apparatus, ordetection apparatus during the deposition and identification processes.In one embodiment, the time between deposition of analyte on thesubstrate and identification is as short as 10 seconds, under conditionsthat the sample is illuminated continuously by the illumination source.In another embodiment, the particle sample identification occurs aftersample deposition. In another embodiment, a background level measurementof the sample substrate is made followed by sample deposition whichoccurs substantially coincident or prior to identification of thesample.

In another embodiment, system 100 is used in a method of the presentdisclosure provides for deposition and visual observation of a threatagent. In a preferred embodiment, the particle sample deposition issubstantially coincident with visual observation of the particle sample.For the purposes of this application, the term substantially coincidentmeans that sample deposition occurs on approximately the same timeperiod as sample observation, ideally without the requirement that thesample substrate be re-positioned relative to the deposition apparatus,or detection apparatus during the deposition and observation processes.In one embodiment, the time between deposition of analyte on thesubstrate and observation is as short as 10 seconds, under conditionsthat the sample is illuminated continuously by the illumination source.In another embodiment, the particle sample observation occurs aftersample deposition. In another embodiment, a background level measurementof the sample substrate is made followed by sample deposition whichoccurs substantially coincident or prior to observation of the sample.

System 100 may operate in a trigger mode or an identification mode. Thetrigger mode detects the presence of a threat agent and the absence of athreat agent. The trigger mode has a trigger time period. The triggertime period may range from a fraction of a second to about 60 seconds.At high concentrations of a threat agent, the trigger time period may besubstantially instantaneous. A one-minute or less trigger cycle timeallows for sampling the environment dynamically to monitor the onset ofthreats and to manage highly variable background conditions.

The identification mode identifies the threat agent and has anidentification time period. In one embodiment, the trigger time periodis less than the identification time period. In another embodiment, theidentification mode is initiated upon detecting the present of thethreat agent in the trigger mode. In another embodiment, theidentification mode is initiated substantially simultaneously upondetecting the present of the threat agent in the trigger mode. Inanother embodiment, an additional amount of sample is accumulated whilethe system is operating in the identification mode.

If the trigger mode is initiated, system 100 changes over to theconfirmation mode under the system software. In the confirmation mode,collection and deposition of additional particles continues during acontinuous 5 minutes interrogation of the sample by the imaging system.No movement of the sample or alignment of sample is needed; thetransition from trigger to confirmation is instantaneous. During theconfirmation time, all particles are examined due to the continuouscollection.

System 100 also includes a processor 128 that determines the mode ofoperation and the identification of the sample. The processor employsdifferent algorithm when system 100 is operation in the trigger mode orconfirmation mode. In one embodiment, the algorithm includes constantfalse alarm rate algorithms. Other algorithms include target testing, aBayesian approach and a matched filter approach based on MahalanobisDistance. Approaches to sample identification are disclosed in: U.S.patent application Ser. No. 10/812,233, filed Mar. 29, 2004 entitledMethod for Identifying Components of a Spectral Analysis; PCTInternational Appl. No. PCT/US05/013036 filed Jul. 14, 2005 entitledMethod and Apparatus for Multimodal Detection; and U.S. ProvisionalPatent Appl. No. 60/688,812, filed Jul. 9, 2005, entitled ForensicIntegrated Search Technology (FIST) each of which is incorporated hereinin its entirety.

To identify the sample, the plurality of spatially resolved spectra,produced by the imaging spectrometer, are compared to at least onereference library spectrum to identify the threat agent. In oneembodiment, the plurality of spatially resolved Raman spectra arecompared to at least one reference Raman library spectrum to identifythe threat agent.

In one embodiment, processor 128 utilizes a target testing for unmixingsignatures and searching the measured mixture spectra relative to thepure component signature library in an automated fashion. Target testingbased spectral unmixing compares mixture spectra against pure componentlibrary spectra by characterizing the mixture space using principalcomponent analysis (“PCA”); ranking the library spectra by quantifyingtheir goodness of fit into the mixture data space; and determining bytarget testing the number and identity of the pure spectra present inthe mixture sample.

With further reference to FIG. 4, the sample was classified as dieselsoot using a Euclidian Distance (ED) matched filter identifieralgorithm. The ED algorithm assumes samples are pure components, whichwas an inaccurate assumption. When the spatially resolved dispersiveRaman spectra are analyzed using a target testing spectral unmixingalgorithm, the mixture sample was correctly classified as beingcomprised of Ova and carbonaceous material, a material class comprisedof DS and humic acid, because of their Raman spectral similarity.

The target testing algorithm includes the following general steps:

-   -   1. Use PCA on the mixture spectra to characterize the mixture        data space.    -   2. Calculate the angle of projection of each library spectrum        with the mixture data space. A dot product of a vector with an        n-dimensional space. A dot product of 1.0 represents a perfect        fit into the data space.    -   3. Rank all library spectra by the angle of projection into the        mixture data space.    -   4. Consider all permutations of the top matches as ranked by        angle. Determine the n most likely candidate pure components.        Generate all possible m component solutions, where m varies from        1 to n and is the number of library spectra in a given solution.    -   5. For each candidate solution calculate the correlation        coefficient; calculate projected library spectra for each set of        m component library spectra (given the known mixture spectra and        the known library spectra). Calculate the correlation        coefficient of each projected library spectrum with the actual        library spectrum. The correlation coefficient used as the        selection criterion is the square root of the sum of squares of        the dot products for each member of a given m component        solution.    -   6. The most probable solution is the one with the highest        correlation coefficient.

The target testing algorithm requires a Raman signature library thatsupports differentiation between threat agents, near neighbors, andclutter independent of agent growth or preparation conditions and samplehistory. Raman spectra of threat agents include certain spectral bandsthat are highly sensitive to growth conditions and others that arerelatively insensitive to growth conditions. The detection andidentification algorithms will focus on spectral bands that maximizeagent discrimination, but minimize sensitivity to growth conditions.This can minimize signature library dependence on unwanted biologicalcontributions to variability.

With further reference to FIG. 1, system 100 includes a depositionsubstrate 110 and substrate positioning mechanism 112. The substrate 110provides for the deposition of a plurality of samples at predeterminedsites. The substrate 110 includes a compact disk (“CD”) configuration orany similar circular or non-circular, substantially flat surface ofmetal or non-metal. The substrate 110 is to enable autonomous focusingof the Raman laser on the substrate. In one embodiment, the substrateincludes a standard audio CD dimensions for the substrate allowing forcollection of approximately 1,800 samples on a standard 120 mm diameterCD. System 100 also includes storage unit capable of holding 25substrates to support 30 days worth of sampling, 43,800 samplescollected in 30 days @ 1,800 samples per substrate. The concept is forthe substrate-disks to be supplied in a cartridge that can be easilyswapped out after 30 days of operation. The storage system includes amarking technique to log the archived samples for conditions at thepoint of data collection e.g., time, date, sensor settings, and forsample relocation. The substrate positioning device 112 includes amotion stage having two degrees of freedom, directional linear motionand rotationally variable. The motion of the substrate positioningdevice 112 generates sample deposits in spiral tracks, circularconcentric tracks, or in linear tracks. The substrates are stored in astorage system designed to protect the resilience of the depositionspots to mechanical shock/vibration, humidity, and other physicochemicalagents that might degrade their stability.

Examples

FIG. 5 illustrates the estimated sensitivity of Raman imagingspectroscopy detection technology. The plot compares system 100estimated signal to noise (“SNR”) vs. bio-aerosol concentration for bothtrigger and confirmation detection modes. The estimations were madeusing a ChemImage Raman system performance model.

As shown in FIG. 5, increasing the time for detection (“T_(d)”) resultsin improved SNR, which enables improved detection sensitivity. Intrigger detection mode, we estimate a LOD of −800 particles per liter(PPL) cells is achievable. In confirmation detection mode, a LOD of 100PPL cells is achievable. LOD is defined as the minimum bio-threatconcentration detectable, in a reproducible manner, at a probability ofdetection (“Pd”) greater than 90%, at the specified T_(d) and a definedprobability of false alarm (“P_(fa)”). The acceptable false alarm rateis determined by operational requirements. However, we have assumed thatin trigger detection mode (T_(d)−30 secs, P_(d)>90%), an acceptablelevel of false alarms is 5/day (P_(fa)−1.7×10³). In confirmation mode(T_(d)−300 secs P_(d)>90%), the required P_(fa) is 1/month(P_(fa)−1.1×10⁴). The calibration curves shown in FIG. 5 are generatedusing a Raman detection SNR performance model.

The present disclosure may be embodied in other specific forms withoutdeparting from the spirit or essential attributes of the invention.Accordingly, reference should be made to the appended claims, ratherthan the foregoing specification, as indicated the scope of thedisclosure. Although the foregoing description is directed to thepreferred embodiments of the disclosure, it is noted that othervariations and modification will be apparent to those skilled in theart, and may be made without departing from the spirit or scope of thedisclosure.

1. A method, comprising: depositing a sample of a threat agent onto asubstrate; illuminating the threat agent via an illumination sourcealong an optical path with a plurality of photons to thereby producephotons transmitted, reflected, emitted or Raman scattered by the threatagent; collecting, via an optical system, elastic scatter photonsproduced by the threat agent and at least one of photons transmitted,reflected, emitted or Raman scattered by the threat agent, wherein saidillumination source is located along an optical path, and said substrateis located along a plane wherein the optical path is at an angle otherthan 90° with respect to the substrate plane and extending a depth offield of the optical system by passing at least one of the followingthrough a phase mask: elastic scattered photons, and photonstransmitted, reflected, emitted or Raman scattered by the threat agent.2. The method of claim 1, further comprising: identifying the threatagent based at least in part on an output of the phase mask.
 3. Themethod of claim 2, wherein identifying the threat agent comprises:analyzing the elastic scattered photons using elastic scattering imagingto form an image of the threat agent; and analyzing at least one ofphotons transmitted, reflected, emitted or Raman scattered by the threatagent using spectroscopic imaging to produce a plurality of spatiallyresolved spectra.
 4. The method of claim 3, wherein analyzing at leastone of transmitted photons, reflected photons, emitted photons or Ramanscattered photons comprises operating in at least one of the followingspectral ranges to produce a plurality of spatially resolved spectra:near infrared; ultraviolet (UV); and mid-infrared.
 5. The method ofclaim 3, wherein analyzing at least one of transmitted photons,reflected photons, emitted photons or Raman scattered photons comprisescollecting images based on one of the following: near infrared imaging;ultraviolet (UV) imaging; mid-infrared imaging; Raman scatter imaging;fluorescence; photoluminescence; chemiluminescence; andelectroluminescence imaging; wherein the near infrared imaging,ultraviolet (UV) and mid-infrared imaging are operated in one of thefollowing transmittance mode and reflectance mode.
 6. The method ofclaim 5, wherein the spectroscopic imaging is performed in conjunctionwith one of the following polarized light microscopy and differentialinterference contrast imaging.
 7. The method of claim 3, whereinanalyzing at least one of photons transmitted, reflected, emitted orRaman scattered by the threat agent further comprises passing said atleast one of photons transmitted, reflected, emitted or Raman scatteredthrough a filter selected from the group consisting of a tunable filter,a band pass filter, a liquid crystal tunable filter, an interferometer,an acousto optic tunable filter and a dispersive optical device, toproduce the plurality of spatially resolved Raman spectra.
 8. The methodof claim 7, wherein the analyzing the at least one of photonstransmitted, reflected, emitted or Raman scattered by the threat agentfurther comprises passing the at least one of photons transmitted,reflected, emitted or Raman scattered, through one of the following: aline scan spectrometer to produce a plurality of spatially-resolvedRaman spectra arising from a sample volume illuminated by theillumination source; a multi-point spectrometer to produce a pluralityof spatially-resolved Raman spectra arising from a sample volumeilluminated by the illumination source; a single point spectrometer toproduce a plurality of spatially-resolved Raman spectra arising from asample volume illuminated by the illumination source; an area imagingspectrometer to produce a plurality of spatially-resolved Raman spectraarising from a sample volume illuminated by the illumination source; anda point spectrometer to produce a single Raman spectrum arising from asample volume illuminated by the illumination source.
 9. The method ofclaim 2, wherein said depositing the sample of the threat agent onto thesubstrate occurs substantially coincident with said identifying thethreat agent on the substrate.
 10. The method of claim 2, wherein saiddepositing the sample of the threat agent onto the substrate occursprior to said identifying the threat agent on the substrate.
 11. Themethod of claim 2, further comprising: identifying a background level ofthe substrate followed by said depositing the sample of the threat agentonto the substrate wherein said identifying the threat agent on thesubstrate occurs after said depositing the sample of the threat agent.12. The method of claim 2, further comprising: identifying a backgroundlevel of the substrate followed by said depositing the sample of thethreat agent onto the substrate wherein said identifying the threatagent on the substrate occurs substantially coincident in time with saiddepositing of the sample of the threat agent onto the substrate.
 13. Themethod of claim 1, wherein the threat agent is a hazardous agentselected from the group consisting of a bacterium, virus, protozoan,biological toxin, fungus, a chemical agent, a radiological material andan explosive material.
 14. The method of claim 13, wherein the hazardousagent is a chemical agent.
 15. The method of claim 13, wherein thehazardous agent is a biological toxin.
 16. The method of claim 13,wherein the hazardous agent is a microorganism.
 17. The method of claim13, wherein the hazardous agent is a bacterium.
 18. The method of claim13, wherein the hazardous agent is a protozoan.
 19. The method of claim13, wherein the hazardous agent is a virus.
 20. The method of claim 1,wherein the threat agent is selected from the group consisting of anairborne particulate matter or aerosol matter.
 21. A system, comprising:means for depositing a sample of a threat agent onto a substrate; anillumination source for illuminating the threat agent along an opticalpath with a plurality of photons to thereby produce photons transmitted,reflected, emitted or Raman scattered by the threat agent; an opticalsystem for collecting elastic scatter photons produced by the threatagent and at least one of photons transmitted, reflected, emitted orRaman scattered by the threat agent, wherein said illumination source islocated along an optical path, and said substrate is located along aplane wherein the optical path is at an angle other than 90° withrespect to the substrate plane; a phase mask for extending a depth offield of the optical system by passing at least one of the followingthrough the phase mask: elastic scattered photons, and photonstransmitted, reflected, emitted or Raman scattered by the threat agent.